Primate Cornea Research Articles

Deficiency of SECTM1 impairs corneal wound healing in aging.

The corneal epithelium is the outermost transparent barrier of the eyeball and undergoes continuous self-renewal by limbal stem cells (LSCs) during its lifetime; however, the impact of aging on LSCs remains largely unknown. Here, we showed that the healing ability of the cornea in elderly macaques (Macaca fascicularis) was significantly decreased compared to that of younger macaques. This delayed wound closure accompanied a disordered cell arrangement and corneal opacity. A novel cytokine, Secreted and Transmembrane 1 (SECTM1), was found to facilitate corneal healing and was upregulated in young macaques upon wounding. Mechanistically, SECTM1 is essential for LSC migration and proliferation, and may partially function through Cell Division Cycle Associated 7 (CDCA7). Notably, the topical application of SECTM1 to aged wounded corneas dramatically promoted re-epithelialization and improved corneal transparency in both mice and macaques. Our work suggests that aging may impair the expression of healing response factors and injury repair in non-human primate corneas, and that SECTM1 application could potentially benefit corneal wound healing in clinical treatment.

Micro-optical coherence tomography for high-resolution morphologic imaging of cellular and nerval corneal micro-structures.

We demonstrate the highest resolution (1.5×1.5×1 µm) micrometer optical coherence tomography (µOCT) imaging of the morphologic micro-structure of excised swine and non-human primate corneas. Besides epithelial, stromal, and endothelial cell morphology, this report focuses on investigating the most peripheral corneal nerve fibers, the nerve fibers of the subbasal plexus (SBP). Alterations of SBP nerve density and composition are reportedly linked to major neurologic disorders, such as diabetic neuropathy, potentially indicating earliest onsets of denervation. Here, the fine, hyperreflective, epithelial nerve structures located just above Bowman's membrane, are i) visualized using our µOCT prototype, ii) validated by comparison to fluorescence confocal microscopy (including selective immunohistochemical staining), and iii) segmented using state-of-the-art image processing. Here, we also introduce polarization sensitive (PS) µOCT imaging, demonstrating, to the best of our knowledge, the highest resolution corneal PS-OCT scans reported to date.

Ocular hypotensive activity of BOL-303259-X, a nitric oxide donating Prostaglandin F2α agonist, in preclinical models

The aim of the study was to investigate the ocular hypotensive activity of a nitric oxide (NO)-donating latanoprost, BOL-303259-X, following topical administration. The effect of BOL-303259-X (also known as NCX 116 and PF-3187207) on intraocular pressure (IOP) was investigated in monkeys with laser-induced ocular hypertension, dogs with naturally-occurring glaucoma and rabbits with saline-induced ocular hypertension. Latanoprost was used as reference drug. NO, downstream effector cGMP, and latanoprost acid were determined in ocular tissues following BOL-303259-X administration as an index of prostaglandin and NO-mediated activities. In primates, a maximum decrease in IOP of 31% and 35% relative to baseline was achieved with BOL-303259-X at doses of 0.036% (9 μg) and 0.12% (36 μg), respectively. In comparison, latanoprost elicited a greater response than vehicle only at 0.1% (30 μg) with a peak effect of 26%. In glaucomatous dogs, IOP decreased from baseline by 44% and 10% following BOL-303259-X (0.036%) and vehicle, respectively. Latanoprost (0.030%) lowered IOP by 27% and vehicle by 9%. Intravitreal injection of hypertonic saline in rabbits increased IOP transiently. Latanoprost did not modulate this response, whereas BOL-303259-X (0.036%) significantly blunted the hypertensive phase. Following BOL-303259-X treatment, latanoprost acid was significantly elevated in rabbit and primate cornea, iris/ciliary body and aqueous humor as was cGMP in aqueous humor. BOL-303259-X lowered IOP more effectively than latanoprost presumably as a consequence of a contribution by NO in addition to its prostaglandin activity. The compound is now in clinical development for the treatment of glaucoma and ocular hypertension.

Histomorphometric Profile of the Corneal Response to Short-term Reverse-geometry Orthokeratology Lens Wear in Primate Corneas

To investigate the histological changes in primate cornea induced by short-term overnight orthokeratology (OK). Nine young adult primates were used. One animal served as negative control. The remaining 8 animals wore reverse-geometry OK lenses for periods of 4, 8, 16, and 24 hours on 1 eye with the other eye as control. Central and midperipheral corneal thickness, as well as ultrastructural changes in corneal epithelium, stroma and endothelium in response to OK lenses, were evaluated. OK significantly reduced the thickness of the central cornea in all treatment groups. The central corneal thinning was both stromal and epithelial in origin. Substantial midperipheral corneal thickening was seen in 16-hour and 24-hour lens-wear groups and this effect was both stromal and epithelial in origin as well. Histology evidence indicated the primary epithelial response in the central cornea was compression of cells that resulted in wing cells becoming shorter and basal cells being squatted rather than lost or migration of cell layers. These pronounced cell shape changes occurred without compromising the structural integrity of the desmosomes. The thickened corneal epithelium has normal cell layers. The squamous cells have larger surface sizes and are composed of oval instead of flattened nuclei. This implied delayed surface cell exfoliation at the thickened midperipheral epithelium. Physical presence of OK lens over the cornea did not influence the microstructures of microvilli and microplicae, endothelium, and collagen distribution. The primate cornea, particularly the corneal epithelium, responds rapidly to the application of reverse-geometry OK lenses with significant epithelial cell shape alterations with short-term OK lens wear. This finding suggests that the corneal epithelium is moldable in response to the physical forces generated by the OK lenses.

Spatial mapping of collagen fibril organisation in primate cornea—an X-ray diffraction investigation

New insights are presented into the collagenous structure of the primate cornea. Wide-angle X-ray diffraction was used to map the fibrillar arrangement and distribution of collagen over three common marmoset corneas. The maps provide a point of reference to help interpret data from pathological corneas or primate models of refractive surgery. The results herein disclose a circum-corneal annulus of highly aligned collagen, 0.5–1.5 mm wide, where the cornea and sclera fuse at the limbus; a feature similar to that observed in human tissue. As in humans, the annulus is not uniform, varying in width, fibril angular spread, and collagen density around its circumference. However, more centrally the marmoset cornea exhibits a preferred lamella orientation in which proportionally more fibrils are oriented along the superior–inferior corneal meridian. This observation is in striking contrast with the situation in human cornea, where there is an orthogonal arrangement of preferentially aligned fibrils. Investigation of a further 16 corneas confirmed that approximately 33% (±1%) ( n=76) of fibrils in the central marmoset cornea lie within a 45° sector of the superior–inferior meridian. Implications for the mechanical and optical properties of the cornea are discussed.

Morphological and Morphometric Aspects of Primate Cornea: A Comparative Study with Human Cornea

Morphological and Morphometric Aspects of Primate Cornea: A Comparative Study with Human Cornea

Quantification of real-time confocal images of the human cornea

Real-time confocal microscopy was used to obtain images of the surface cells of the cornea in vivo in human subjects and in non-human primates. The surface cells represent the barrier between the tear layer and the internal cellular environment and as such, the state of these cells is indicative of the health of the cornea. In our study, confocal microscopy of the surface cells revealed prominent, centrally located nuclei. With the use of a series of image analysis procedures, the nuclei were located automatically and distances to the nearest neighbors were determined. Comparison of these procedures in 8 human corneas and 1 non-human primate cornea showed that unaided computer analysis of the surface cells was as accurate as manual location of the cell nuclei. The distribution of nearest-neighbor distances was found to be best fitted by a gamma distribution. Simulation of a condition marked by loss of surface cells demonstrated that the alpha (shape) and beta (scale) parameters could be used to compare the distribution of nearest-neighbor distances. Thus, confocal microscopy coupled with these image analysis and statistical procedures could provide an objective, quantitative approach to monitoring the epithelial barrier under clinical and experimental conditions, for example during post-surgical or post-traumatic healing or in the evaluation of the efficacy of topical therapeutic agents.

Quantitative Histological Studies of Primate Corneas After Excimer Laser Photorefractive Keratectomy

To evaluate histological changes in the primate cornea after excimer laser photorefractive keratectomy (PRK) and to correlate them with clinical observations. Sixteen African green monkey (Cercopithecus aethiops sabaeus) corneas were examined by light and transmission electron microscopy 6 weeks to 18 months after 1.5- or 3-diopter PRK. All specimens had a smooth stromal surface surrounded by a smooth, centrally tapered Bowman's layer. Epithelial thickness appeared to peak 12 months after PRK. The trend was for the epithelium to be thickest in the central-treated area; this phenomenon was more obvious in the 3-diopter-treated corneas. The numbers of activated keratocytes beneath the treated zone peaked at 4 months and decreased thereafter, while the numbers in the untreated areas decreased in the first 2 months after surgery, increased by 4 months, and did not change thereafter. Regenerated basal lamina averaged 86% intact over ablated areas; thickness was normal and no duplications were seen. Overall, the density of hemidesmosomes was significantly less in ablated areas compared with unablated areas. These findings support the relationship between clinical observations of corneal haze after PRK, reestablishment of the epithelial cell layer, and the potential for stromal remodeling by active fibroblastic keratocytes beneath the ablation zone. Overall, quantification of several morphological parameters indicated that the values for the treated zone tended, with time, to approach those of the untreated cornea after PRK.

Long-term effect of erbium-YAG laser (2.9 ?m) on the primate cornea

An erbium-YAG (2.9 microns) laser was used in the primate eye to irradiate the anterior surface of the cornea in one group and the midstroma in another group after temporary lamellar keratectomy. The eyes were observed clinically up to a period of eight months, when the animals were sacrificed. The initial opacification of the cornea caused by the erbium laser application gradually disappeared over the observation period and was replaced by a faint haze. The healing process was faster in Group II (midstromal laser ablation). Some degree of flattening of the cornea after laser ablation was seen in both groups by photokeratoscopy. At the end of the observation period, the irradiated corneas demonstrated normal appearing corneal structure without significant damage to the corneal epithelium, stroma, or endothelial cells. Further experimental studies are needed to improve the laser delivery system and reduce the degree of corneal damage produced by long laser pulses.

Corneal Damage in Photokeratitis???Why Is It So Painful?

The primate cornea was examined ultrastructurally subsequent to ultraviolet radiation (UVR) (300 nm) exposures of 0.08 and 0.225 J/cm2. The lower exposure caused significant, but incomplete, destruction of the epithelium and mild edema of the posterior stroma. The higher exposure resulted in complete destruction of the epithelium, significant stromal swelling, and some decomposition of endothelial cells. Widespread intracellular edema of the endothelium was also noted with the higher exposure. The ultrastructural examination subsequent to these UVR exposures also helps to explain why UV-induced keratitis is so painful. The 0.08 J/cm2 exposure left the epithelial and subepithelial-stromal nerve plexuses intact, whereas the 0.225 J/cm2 exposure destroyed the epithelial axons but spared the subepithelial ones. These results suggest that the pain associated with UV keratitis results from the combined effect of epithelial cell loss and sparing of the subepithelial axons.

One-Year Refractive Results of Central Photorefractive Keratectomy for Myopia in the Nonhuman Primate Cornea

Photorefractive keratectomy for the correction of myopia was performed in 32 eyes of 16 green monkeys. The corneas healed satisfactorily, with normal formation of basal lamina and hemidesmosomal attachments visible in 14-week histologic specimens. No recurrent erosions were observed clinically. After a transient period of faint haze, all corneas were clear at 17 weeks and remained clear through the 1-year follow-up. In terms of accuracy, all corneas demonstrated a significant flattening compared with preoperative values, but no significant difference was seen between the groups with different intended corrections (1.5 and 3 diopters). The changes in corneal shape stabilized by 17 weeks, as measured by keratometry. The clinical results suggest that mechanical removal of the epithelium is preferable to laser ablation of the epithelium. Overall, the results demonstrate that excimer laser ablation of the corneal stroma can produce a stable diptric change in the primate cornea with good healing and long-term corneal clarity.

Scanning slit delivery system

We describe the structure and function of a delivery system designed for use with an argon fluoride excimer laser for corneal surgery. The basic principle of the delivery system is to shape the laser beam with a slit mask to a defined configuration. The image of the slit is moved across the cornea in a mathematically defined pattern to ablate a given amount of tissue, creating a new anterior radius of curvature for the cornea. The location of the beam on the cornea is determined by a rotating dove prism and a translating spherical lens, each of which is controlled by computer-regulated servomotors. The system is versatile and can be used for laser myopic or hyperopic keratomileusis, for correction of astigmatism by surface ablation, for creation of radial or transverse linear cuts, for excision of corneal buttons, and for uniform lamellar corneal smoothing procedures. The delivery system is currently used in the laboratory for the ablation of nonhuman primate corneas.

Growth Factors and Corneal Epithelium

Regeneration of corneal epithelium following injury is essential for visual rehabilitation. A limited number of approaches are available for treating patients who fail to heal epithelial injuries adequately. The presence of specific receptors for epidermal growth factor (EGF) on epithelial cells suggests that this potent mitogen may play a role in normal epithelial wound healing. Topical application of biosynthetic human EGF significantly accelerated epithelial regeneration in primates following epikeratophakia surgery. Epidermal growth factor alone and with fibronectin accelerated epithelial regeneration of rabbits following mild alkali burns. Since prolonged exposure of cells to EGF is necessary to induce mitosis, the dynamics of EGF in the eye and with various lenses was studied. When applied in methylcellulose-based eye drops, 90% of the EGF was lost from tear film within 10 min, while a small amount (10%) remained associated with conjuctival tissue. Soft contact lenses or epikeratophakia lenticles took up substantial amounts of EGF (50 μg) and released 85% of the EGF within 24 h, with a half-life of 4 h in vitro. Epidermal growth factor did not diffuse through corneas or lenticles and did not promote epithelial down-growth along sutures in primate corneas. These results suggest that biosynthetic growth factors may be useful in the treatment of some epithelial injuries.

Ultrastructural analysis of corneal exposure to UV radiation.

The primate cornea was exposed to 300 nm UVR with five levels of radiant exposure from 0.08 to 0.6 Jcm-2. All cellular layers of the cornea were damaged at the 0.08 Jcm-2 exposure, and damage became more severe as the exposure level was increased. The corneal cells showed variable response in that essentially normal cells were found among damaged cells. Eight days post-exposure using the 0.6 Jcm-2 level, the epithelium had regained its normal thickness and was populated largely by normal appearing cells; however, the stroma showed damaged keratocytes and the loss of keratocytes. The corneal basement membranes (the epithelial basement membrane and the posterior limiting lamina) and the anterior limiting lamina were not damaged at any exposure level except for an isolated area along the epithelial basement membrane in one cornea. Therefore, one is lead to conclude that basement membranes are unaffected by UVR. The endothelium continued to demonstrate the loss of mitochondria, endoplasmic reticulum and some vacuoles at 8 days after exposure. However, the endothelium appeared to have resumed its physiological function as demonstrated by the reduced stromal oedema. This research gives the first complete description of UV-B induced corneal damage and repair of the full, in-depth cornea of the primate using the EM.

Growth Factors and Corneal Epithelium

Regeneration of corneal epithelium following injury is essential for visual rehabilitation. A limited number of approaches are available for treating patients who fail to heal epithelial injuries adequately. The presence of specific receptors for epidermal growth factor (EGF) on epithelial cells suggests that this potent mitogen may play a role in normal epithelial wound healing. Topical application of biosynthetic human EGF significantly accelerated epithelial regeneration in primates following epikeratophakia surgery. Epidermal growth factor alone and with fibronectin accelerated epithelial regeneration of rabbits following mild alkali burns. Since prolonged exposure of cells to EGF is necessary to induce mitosis, the dynamics of EGF in the eye and with various lenses was studied. When applied in methylcellulose-based eye drops, 90% of the EGF was lost from tear film within 10 min, while a small amount (10%) remained associated with conjuctival tissue. Soft contact lenses or epikeratophakia lenticles took up substantial amounts of EGF (50 micrograms) and released 85% of the EGF within 24 h, with a half-life of 4 h in vitro. Epidermal growth factor did not diffuse through corneas or lenticles and did not promote epithelial downgrowth along sutures in primate corneas. These results suggest that biosynthetic growth factors may be useful in the treatment of some epithelial injuries.

Electron Microscopic Study of Intrastromal Hydrogel Implants in Primates

Three corneas with intrastromal hydrogel implants (surfilcon A) were removed from Green monkey eyes by penetrating keratoplasry and examined by light microscopy, as well as scanning and transmission electron microscopy, in order to assess the tolerance of the primate cornea for this type of synthetic plastic. Placement of these implants in the posterior cornea appears to increase the amount of protrusion into the anterior chamber, which can also be seen clinically on slit-lamp biomicroscopy. Some physiological stress to the cornea was indicated by abnormalities above and below the implant, including thinned epithelium, irregular cell shapes, and vacuolations. It appears that a basement-membrane-like material is produced by keratocytes adjacent to the implant-stroma interface, and that this material fills the spaces and provides some physical continuity between the plastic and the corneal tissue. No inflammatory reaction was seen around the implants, but further long-term studies are necessary to ensure compatibility between the cornea and the implant.

Myopic Hydrogel Keratophakia

Myopic hydrogen keratophakia offers several potential advantages over existing forms of corneal refractive surgery. Because no tissue lathing is required, neither donor tissue nor complex lathing equipment is necessary. Theoretically, hydrogen lens implants with precisely determined and verifiable parameters can be obtained in limitless supply. The surgical effect can be either modified or reversed, if clinically indicated. In this preliminary report, myopic hydrogel implants, with powers between -5 diopters and -20 diopters, were placed in nonhuman primate corneas. Corneascopic analysis showed significant flattening in all instances, and about 70% of the anticipated correction was actually observed. Operative complications were infrequent and generally resulted from traumatic wound dehiscence. Myopic hydrogel keratophakia has tremendous clinical potential, but requires further experimental testing as to safety and efficacy.

Alloplastic Epikeratophakia for the Correction of Aphakia

SUMMARY Plastic implants were combined with epikeratophakia grafts on the primate cornea. Implants in intrastromal pockets survived but did not substantially increase refractive powers. When implants were placed simultaneously with lyophilized epikeratophakia grafts, necrosis and loss of the graft ensued. Alloplastic implants survived on the cornea only if the overlying tissue contained living keratocytes, i.e., fresh donor tissue grafted over the implant or established lyophilized grafts in which the peripheral scar was opened slightly and the implant slipped between the graft and the cornea. Advantages of easily replaced implants include the ability to accommodate changing refractive needs, as for infants with monocular aphakia.

Observations on the morphology of the developing primate cornea: Epithelium, its innervation and anterior stroma

AbstractA survey is made of some ultrastructural features of the developing cornea of Macaca mulatta. The observations are confined to the anterior central area, starting with the lens vesicle stage and progressing through midgestation, when the morphologic characteristics of the cornea are fully established. Subepithelial filaments and some partially aggregated collagen fibrils are present in the earliest embryo and are of a size and appearance similar to those in the future vitreous cavity. Epithelial secretory activity points to, but does not prove direct contribution to the deposition of the acellular matrix components beneath it. No trace of a structured, orthogonal collagenous stroma can be visualized. The primitive endothelium forms prior to the fibroblast invasion of the distended filamentous matrix. Bowman's layer has undoubted epithelial contributions. Its aggregated collagen fibrils have approximately the same diameter as those of the anterior stroma. Intraepithelial appearance of single nerve fibers and fascicles takes place during the first trimester of gestation, as soon as the two continuous epithelial layers are formed. Terminal areas approach closely to the basal cell's nucleus, without touching it. The plasmalemma of the invaginating nerve fiber is surrounded by that of the epithelial cell in a mesaxon‐like manner, with occasional gap junctions uniting adjoining neural and epithelial cell membranes. The fetal neurites contain microtubules, some clear vesicles and dense vacuoles resembling those of mature monamine and non‐monamine neurons. Mitochondria are small and compact, their presence indicating a high rate of metabolic activity in the immature terminal area.

Endothelial wound repair in primate cornea

In vivo transcorneal freezing was used to destroy two-thirds of the endothelial cells in monkey cornea and the sequential process of repair was studied by biomicroscopy, pachometry, scanning and transmission electron microscopy. The freezing injury resulted in complete denudation of cells from Descemet's membrane in the area of the probe, and the corneas became grossly edematous and doubled in thickness during the first 24 hr. Repopulation of the denuded area was complete by 7–9 days and appeared to be due to the enlargement and migration of the marginal endothelial cells. Corneal thickness decreased after the endothelial layer was re-established, but after stabilization remained at least 0·1 mm above control values. Similarities between the pattern of endothelial repair in the primate and of endothelial decompensation (due to age or injury) in the human suggest that this may be a better model for studies of endothelial regeneration than is the rabbit cornea.